Mammary development occurs in two distinct phases, an initial phase of modest growth (embryonic to puberty) and a later phase of robust expansion. Not surprisingly, studies on the early, relatively "quiescent" phase are greatly overshadowed by the latter. Nevertheless, changing conditions during the early phase have important consequences on later mammary development and carcinogenic potential. This was clearly identified when it was found that estrogenic exposure in 5-day estrogen-treated newborn mice resulted in a proliferative response, but one that occurred later at puberty. Paracrine estrogen receptor (ER1) stimulation elevates growth hormone-induced production and release of insulin-like growth factor 1 (IGF-I) and thereby induces an epithelial proliferative response, but the reason for the delay is unclear. Our published and preliminary results and those of others raise the possibility that the homeobox transcription factor HoxC6 may underlie this delayed response: 1) Mammary ductal trees in our HoxC6 deficient animals display a profound failure to undergo postnatal expansion;2) In normal animals, HoxC6 is expressed in epithelium and stroma and stromal expression is repressed by estrogen;3) HoxC6 is a direct transcriptional repressor of IGF binding protein 3 (IGFBP3), and IGFBP3 upregulation occurs in HoxC6-downregulated mammary epithelial cell lines and HoxC6 null mammary glands. In the latter case, we also observed downregulation of matrix metalloproteinases (MMPs) and upregulation of inhibitors of MMPs, indicating IGFBP3 stabilization. This would suggest that neonatal exposure to estrogen would indeed stimulate production of IGF-I (via ER1), but also repress HoxC6, resulting in an increase in IGFBP3 synthesis and stability. The presence of IGFBP3 would sequester and stabilize IGF-I in the stroma and blunt an immediate epithelial response. At puberty, ER1 hyper-stimulation would destabilize IGFBP3, thereby releasing stores of IGF-I for excessive growth/survival and ductal expansion. Excessive expansion of undifferentiated mammary epithelial stem cells being a link to risk. Our overall hypothesis is that HoxC6 mediates the estrogen-induced delay in mammary epithelial cell proliferation by de-repressing IGFBP3 expression, blunting IGF-I stromal-to-epithelial signaling in the early phase. This will be tested experimentally in one Specific Aim. The goal of AIM 1 is to define the mechanism by which HoxC6 modulates IGF-I stromal-to-epithelial signaling in the regulation of mammary epithelial growth/survival &branching. This will be accomplished using: 1) cell type-specific cultures;2) whole organ cultures;and 3) in vivo models. In all studies, the impacts of estrogen exposure and HoxC6 expression status in mammary stroma and epithelium will be measured regarding IGF-I signaling, gene expression (IGF-I, HoxC6, IGFBP3, FGFR2) and cellular characteristics of growth/survival, migration, differentiation and branching. A practical goal of this study is to utilize HoxC6 gene expression levels in vitro as a surrogate for the assessment of anthropogenic and natural compounds that impact on postnatal mammary epithelial carcinogenic potential. PUBLIC HEALTH RELEVANCE: Early menarche and breast development are risk factors for breast cancer, which is the second leading cause of cancer-related deaths in women, and early exposure to natural and anthropogenic estrogens can profoundly sensitize the mammary gland to early development and cancer. In this study, we will test our model that early exposure to estrogens stimulate production of excess growth factor that is sequestered and stabilized and thereby elicits early menarche and hyper-stimulation of mammary growth/survival and expansion. The practical application of this work is toward the development of high throughput identification of bioactive compounds that promote or prevent breast carcinogenesis.